Novel carbazole sulfonamide derivatives of antitumor agent: Synthesis, antiproliferative activity and aqueous solubility

Article abstract of DOI:10.1016/j.bmcl.2016.11.068

The current optimization of IG-105 (3) on the carbazole-ring provided a series of new carbazole sulfonamides derivatives 13a–13m. All of the compounds have been evaluated against HepG2 cells (hepatoma cancer) for antiproliferative activity. Compounds that showed activity better or comparable to that of 3 versus HepG2 were evaluated against MCF-7 (breast cancer), MIA PaCa-2 (pancreatic cancer), and Bel-7402 (hepatoma/liver cancer) for antiproliferative activity. Of the seven compounds selected for further study five (13b, 13g, 13j, 13k and 13l) were found to give IC₅₀values against the four cell lines comparable to those for 3. Two compounds (13f and 13i) were more active than 3 and their activity against HepG2 and MCF-7 (IC₅₀:0.01–0.07?μM) approached that of the positive controls podophyllotoxin (podo) and CA-4. Most of compounds showed aqueous solubility (0.11–19.60?μg/mL at pH 7.4 and 2.0) better than 3. These promising results warrant further development of new compounds 13f and 13i as potential potent antitumor drug candidates.

Full text of DOI:10.1016/j.bmcl.2016.11.068

Accepted Manuscript
Novel Carbazole Sulfonamide Derivatives of Antitumor Agent: Synthesis, An-
tiproliferative Activity and Aqueous Solubility
Lianqi Sun, Yanbin Wu, Yonghua Liu, Xiaofang Chen, Laixing Hu
PII:
S0960-894X(16)31214-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.11.068
BMCL 24463
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
19 September 2016
9 November 2016
22 November 2016
Please cite this article as: Sun, L., Wu, Y., Liu, Y., Chen, X., Hu, L., Novel Carbazole Sulfonamide Derivatives of
Antitumor Agent: Synthesis, Antiproliferative Activity and Aqueous Solubility, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.11.068
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Novel Carbazole Sulfonamide Derivatives of Antitumor Agent: Synthesis,
Antiproliferative Activity and Aqueous Solubility
a
a
a
a*
a*
Lianqi Sun , Yanbin Wu , Yonghua Liu , Xiaofang Chen , Laixing Hu
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences &
Peking Union Medical College, Beijing 100050, People’s Republic of China
Corresponding author. Tel. /fax: +86 10 63132113.
E-mail address: hulaixing@hotmail.com (L.X. Hu); chenchemistry@hotmail.com
X.F. Chen)
a
*
(
Abstract:
The current optimization of IG-105 (3) on the carbazole–ring provided a series of new
carbazole sulfonamides derivatives 13a-13m. All of the compounds have been
evaluated against HepG2 cells (hepatoma cancer) for antiproliferative activity.
Compounds that showed activity better or comparable to that of 3 versus HepG2 were
evaluated against MCF-7 (breast cancer), MIA PaCa-2 (pancreatic cancer), and
Bel-7402 (hepatoma/liver cancer) for antiproliferative activity. Of the seven
compounds selected for further study five (13b, 13g, 13j, 13k and 13l) were found to
give IC50 values against the four cell lines comparable to those for 3. Two compounds
(
(
13f and 13i) were more active than 3 and their activity against HepG2 and MCF-7
IC50：0.01-0.07 M) approached that of the positive controls podophyllotoxin (podo)
and CA-4. Most of compounds showed aqueous solubility (0.11-19.60 µg/mL at pH
.4 and 2.0) better than 3. These promising results warrant further development of
7
new compounds 13f and 13i as potential potent antitumor drug candidates.
1

Keywords: carbazole sulfonamide derivatives; synthesis; antiproliferative activity;
aqueous solubility
Microtubules, as key components of cytoskeleton, play important roles in a series of
cellular processes such as regulation of motility, cell signaling, formation and
maintenance of cell shape, secretion, and intracellular transport. Microtubule-binding
agents (MBAs) are usually divided into 3 categories including taxol, vinblastine and
[
1, 2]
colchicine binding site/domain inhibitors.
Compounds like colchicine and
podophyllotoxin (podo), discovered in the early days irreversibly bind at the
colchicine domain thus inducing significant toxicities and limiting clinical use. The
reversibly binding inhibitors discovered later, like Combretastatin A4 (CA-4, 1), with
effective dose much lower than maximum tolerated dose, have served as clinical
candidates for further development.
<Figure1>
Combretastatin A4 (1), a novel small molecule tubulin binding agent which was
[
3]
isolated from the South African bush willow Combretum caffrum by Pettit et al.,
strongly inhibits the polymerization of tubulin by binding to the colchicine binding
[
4]
site. Potent cytotoxicity for 1 has been observed against a wide variety of cell lines,
including multidrug resistant lines. However, the low water solubility of this
compound has limited its efficacy in vivo. Its water-soluble prodrug，CA-4 phosphate
disodium 2 is now in phase II clinical trials both as a single drug and in combination
[
5]
for anticancer therapy.
It appears to act by a unique irreversible and selective
[
6]
antivascular effect that disrupts immature endothelial cell’s cytoskeleton in vivo.
2

Over the years, a large number of structure-activity relationship (SAR) studies on 1
have been reported by several academic and industrial groups. In brief, it is thought
that the presence of the 3, 4, 5-trimethoxy group on ring A, the cis-orientation of the
two aromatic rings, and the para-methoxy group on ring B are essential for optimal
[
7]
cytotoxic activity. Unfortunately, CA-4 and other olefinic analogues are prone to
isomerize into their inactive trans-forms during storage and administration. To
overcome this problem, varieties of conformationally restricted cis-locked analogues
have been designed through the replacement of the olefinic bond with heterocyclic
moieties. We also were interested in exploring the alteration of the linker and the A,
B-rings; we replaced the cis-olefin linker with a sulfonamide group, the
3
,4,5-trimethoxyphenyl with 2,6-dimethoxylpyridine, the B ring with a carbazole-
[
8]
ring which led to the synthesis IG105 (3).
<Figure 2 >
Our previous studies with 3 have shown a potent activity against human leukemia and
solid tumors in breast, liver, prostate, lung, skin, colon and pancreas with IC50 values
between 0.012 and 0.298 µM. In addition, 3 was shown to inhibit microtubule
assembly by binding at colchicine pocket. Compound 3 shows a potent anticancer
activity in vitro and in vivo and has good safety in mice, however its the poor water
[
9]
solubility limits oral absorbability.
Transforming the structure of 3 so as to
significantly increase the water-solubility and improve the oral bioavailability while
maintaining or increasing the anti-tumor activity is the goal of this work. The
compounds selected for synthesis have various substitutions on the carbazole-ring
3

which will also expand the SAR of this series of carbazole sulfonamides. Thus, we
focused on polar or ionizable groups on the 6- and 7-positions of the carbazole-ring
which are expected to provide activity comparable to 3. Furthermore, the presence of
R substituent(s) which can serve as H-bond acceptors or donors is expected to be
favorable for improving aqueous solubility. Therefore, a series of new carbazole
sulfonamides derivatives with different R groups on the carbazole-ring are reported
herein.
As shown Scheme 1, carbazole, a commercially available and inexpensive compound,
was allowed to react with CH I in DMF in the presence of excess NaOH at room
3
temperature to produce 9-methyl-9H-carbazole (4) with a yield 95%. Compound 4
was separately brominated, nitrated, and formylated to afford 11a, 11b, and 11c.
However, 11d and 11e were synthesized by a 4 steps process of nitration, Suzuki
coupling, cyclization to the carbzole and finally methylation starting with
4
-bromobenzaldehyde or 1-Bromo-4-nitrobenzene, respectively.
In Scheme 2, compounds 12a, 12b and 12d were obtained from compounds 11a, 11b
[
10]
and 11d using Gupta’s method.
afforded using the same method from 11c and 11e. Finally, we found that 12c and 12e
could be afforded successfully through 11c and 11e reacting with excess ClSO H at
Intermediates 12a-12e were reacted with 3-amino-2,
-dimethoxypyridine in DMF in the presence of potassium carbonate with stirring at
However, compounds 12c and 12e can’t be
3
[11]
0
6
°C in 70%-80% yields.
room temperature for about 2-3 h to afford 13a-13e in yields of 70%-85%. In addition,
the aldehyde group in 13c and 13e were reduced with NaBH to provide compounds
4
4

1
3g and 13j with a hydroxymethyl substituent in yields of 65% and 70%, respectively.
The aldehydes group in 13c and 13e were oxidized with H (35% in water) and
conc. sulfuric acid in methanol at room temperature to provide the carboxylic acid
3k in yield 71% and the ester 13h in yield 45%. 13e was allowed to react with
2
O
2
1
m-chloroperbenzoic acid (m-CPBA) in DCM at room temperature for about 24 h to
give the corresponding formate ester. Subsequently, the formate ester was hydrolyzed
to afford 13f in 60% yield. Catalytic hydrogenation of 13b or 13d with hydrogen (40
PSI) in the presence of Pd/C (5-10%) in anhydrous methanol afforded 13l or 13i in
7
5% yield. 13k was allowed to react with CH
3
I in DMF in the presence of excess
NaOH at room temperature to produce 13m in 50% yield.
<
<
Scheme 1>
Scheme 2>
All of the target compounds were evaluated against HepG2 cells for antiproliferative
[
12]
activity by the standard sulforhodamine B (SRB) assay.
Seven compounds, which
showed activity versus HepG2 better than or comparable to that of 3, were evaluated
against MCF-7, MIA PaCa-2, and Bel-7402 for antiproliferative activity. The data are
summarized in Table 1 and Table 2. Compounds 13f, 13i were found to have lower
IC50 values against the four cell lines than 3. Against HepG2 and MCF-7 their IC50
values (IC50: 0.01-0.07 µM) approach that of the positive control drugs podo and
CA-4. Compounds 13b, 13g, 13j, 13k and 13l were found to have IC50 values
equivalent to that of 3 against four cell lines. On the other hand, compounds 13a, 13c,
1
3d, 13e and 13m were significantly less potent than 3 against HepG2 cells (IC50:
0
.26µM, 0.26µM, >25µM, 0.22µM and 7.22 µM, respectively). Thus, the current
5

results demonstrated that the R substituent on the carbazole-ring was modifiable and
could greatly affect potency. When the R substituent is either a hydroxyl group or an
amino group the highest antitumor activity is observed.
It is very interesting that compounds 13e, 13f, 13g and 13i which have the R groups at
the 7-position of the carbazole ring generally showed cytotoxic activity greater than
that of compounds 13c, 13k, 13j and 13l with the R group at the 6-position of the
carbazole ring. Interestingly, the most active compounds 13f and 13i (substituted in
the 7-position) exhibited greater potency against four tumor cell lines in vitro, than the
corresponding 6-substituted compounds 13k and 13l.
To contribute to the structural understanding of the activity of these compounds, we
performed docking studies with four compounds at the colchicine binding pocket
using the CDOCKER program in the Discovery Studio 3.0 software and the tubulin
[
13]
crystal structure (PDB code: 4O2B) in comparison with the 3. As shown in Figure
3
, a key amino acid Asn258 potentially can form hydrogen bonds with the same group
of compounds 13f, 13i, 13k, 13l and 3. In addition, 13f, 13i could form an additional
hydrogen bond with amino acid Val238, and similarly 13k, 13l could form an
additional hydrogen bond with amino acid Asp251. These additional H-bonds may
enhance the binding affinity of these four compounds (13f, 13i, 13k, 13l) with tubulin
and subsequently lead to the increase antitumor activity observed. All the calculated
distances between ligands and amino acids is less than 3 Å, and the distance between
hydroxyl group and amino acid is less than the distance between amino group and
amino acid, which may contribute to the explanation for the higher antitumor activity
6

of 13f, 13k (hydroxyl group-substituted) than that of 13i, 13l (amino
group-substituted) .
<Figure 3 >
An important goal of this research was to increase the aqueous solubility of the new
compounds compared to that of 3. To assess this property we measured their aqueous
solubility by using an HPLC/UV method under two conditions: pH = 2.0 and pH =
[
14]
7
.4 as shown in Table 1.
All of the tested compounds, except 13b and 13d, showed
improved solubility at both pH 2.0 and pH 7.4 compared with that of 3. Notably, the
most active compound 13f had aqueous solubility of 0.84µg/mL at pH 2.0 and 1.53
µg/mL at pH 7.4 which represents a 100-fold increase over that of 3. Generally, most
of compounds showed equivalent aqueous solubility under the two conditions, but the
compounds 13i and 13l were more soluble in the acidic solution than neutral one,
presumably because of the presence of the amine group. A comparison of the
6
-substituted and 7-substituted series showed that the 6-substituted compounds were
generally more soluble than the corresponding 7-substituted compounds.
<
Table 1>
Table 2>
<
Following our optimization strategy, 13 novel carbazole sulfonamide derivatives were
synthesized by modifying the R group on the carbazole-ring of 3. Compounds 13b,
1
3g, 13j, 13k and 13l showed significant cytotoxic activity against HepG2、MIA
PaCa-2、MCF-7 and Bel-7402 cell lines with IC50 values at a sub-micromole level
(
IC50: 0.05-0.1 µM) comparable to that of 3. Compounds 13f, 13i were found to have
7

lower IC50 values against the four cell lines than 3. Against HepG2 and MCF-7 the
IC50 values for 13f and 13i (IC50: 0.01-0.07µM) approached those of the positive
controls podo and CA-4. The modifications of the R substituent on the carbazole-ring
provided the following new SAR information: (1) when the R substituent is the
hydroxyl group or the amino group, compounds show the highest antitumor activity
and solubility; (2) the antitumor activity of the compounds with substituted at the
7
-position of carbazole ring ware generally more potent than the activity of the
corresponding compounds with substituted at the 6-position of the carbazole ring.
Furthermore, most of compounds showed aqueous solubility (0.11-19.60 µg/mL at pH
7
.4 and 2.0) better than 3. A comparison of the 6-substituted and 7-substituted series
showed that the 6-substituted compounds were generally more soluble than the
corresponding 7-substituted ones. These promising results for new compounds 13f
and 13i as potential potent antitumor drug candidates warrant further development.
Acknowledgements:
This investigation was supported by Grants (30801427 and 81321004) from the
National Natural Science Foundation of China (NSFC).
References and Notes:
1
2
3
4
5
. Downing, K. H.; Nogales, E. Curr. Opin. Cell Biol. 1998, 10, 16.
. Jordan, M. A.; Wilson, L. Nat. Rev. 2004, 4, 947.
. Pettit, G. R.; Cragg, G. M.; Singh, S. B. J. Nat. Prod.1987, 50, 386.
. Young, S. L.; Chaplin, D. J. Expert Opin. Invest. Drugs. 2004, 13, 1171.
. Mooney C.J.; Nagaiah G.; Fu, P.; Wasman J.K. Thyroid, 2009, 19, 233-240.
8

6
7
. Tozer, G. M.; Kanthou, C.; Baguley, B. C. Nat. Rev.Cancer 2005, 5, 423.
. Chaudhary, A.; Pandeya, S. N.; Kumar, P.; Sharma, P. P.; Gupta, S.; Soni, N,;
Verma, K. K; Bhardwaj, G. Mini-Rev. Med. Chem. 2007, 7, 1186.
8
9
1
. Hu, L. X.; Li, Z. R.; Li, Y.; Qu, J. R.; Ling, Y. H.; Jiang, J. D.; Boykin, D. W. J.
Med. Chem. 2006, 49, 6273.
. Wang, Y. M.; Hu, L. X.; Liu, Z. M.; Boykin, D. W.; Jiang, J. D. Clin. Cancer. Res.
2
008, 14, 6218.
0. Gupta, A. K.; King, S. A.; Lee, E. C.; Morton, H. E.; Plata, D. J.; Pu, Y. M.;
Sharma, P. N. WO 2002024648.
1
1
1. Jarboe, S. G.; Terrazas, M. S.; Beak, P. J. Org. Chem.2008, 73, 9627-9632.
2. The standard sulforhodamine B (SRB) assay. The SRB assay was used for in
vitro anticancer study of the synthesized compounds. The human cancer cells
were inoculated into 96-well microtiter plates at plating densities of 4000-6000
cells/well and were incubated for 24 h. Treatment of the cells with the solutions of
compounds in DMSO was done after 24 h, and watered down in medium to yield
4
different concentrations of 2.5, 5, 10 and 20 µg/ml, while cells that contained no
drugs/sample were used as control and blanks comprised complete medium with
no cells. Incubation of plates for 48 h was followed with addition of the
compounds. Viable cells were fixed to the bottom of each well with cold 50%
trichloroacetic acid, and washed, dried and dyed by SRB. The boundless dye was
detached, while 10 mM Tris base was used for the extraction of protein-bound dye,
and its optical density determination achieved using
a
multi-well
9

spectrophotometer at the wavelength 570 nm. 50% of cell growth inhibition was
calculated.
1
1
3. Prota, A.E., Danel, F., Bachmann, F., Bargsten, K., Buey, R.M., Pohlmann, J.,
Reinelt, S., Lane, H., Steinmetz, M.O. J.Mol.Biol. 2014, 426, 1848-1860.
4. Aqueous Solubility Determination. Solubility was measured separately at pH
7
.4 and pH 2.0 by using an HPLC-UV method. Test compounds were initially
dissolved in DMSO at 10 mg/mL. Ten microliters of this stock solution was
spiked into either pH 7.4 phosphate buffer (1.0 mL) or 0.01 M HCl
(
approximately pH 2.0, 1 mL) with the final DMSO concentration being 1%. The
mixture was stirred for 4 h at r.t., and then concentrated at 3000 rpm for 10 min.
The saturated supernatants were transferred to other vials for analysis by
HPLC-UV. Each sample was performed in triplicate. For quantification, a model
1
200 HPLC-UV (Agilent) system was used with an Agilent TC-C18 column (250
4.6 mm, 5 m) and gradient elution of acetonitrile (ACN) in water, starting
×
with 0% of ACN, which was linearly increased up to 70% over 10 min, then
slowly increased up to 98% over 15 min. The flow rate was 1.0 mL/min and
injection volume was 15 μL. Aqueous concentration was determined by
comparison of the peak area of the saturated solution with a standard curve plotted
peak area versus known concentrations, which were prepared by solutions of test
compound in ACN at 50 μg/mL, 12.5 μg/mL, 3.125 μg/mL, 0.781μg/mL, and
0
.195 μg/mL.
10

Figure1 Structures of CA-4 and CA-4P
11

Figure 2 Structure of IG-105
12

A
B
C
D
Figure3 Predicted binding modes of four compounds (different colors) and 3 (grey) with tubulin,
respectively (PDB code: 4O2B). A: 13f (red); B: 13i (cyan); C: 13k (blue); D: 13l (green).
13

Reagent and conditions: a: CH
HOAC, rt. 0.5h; d: POCl , DMF, 0-80 °C, 3 h; e: NaNO
phenylboronic acid, Pd(PPh , K CO , toluene, reflux, 12 h; g: PPh
,2-dichlorobenzene, reflux, 6 h.
3
I, NaOH, DMF, 4 h; b: NBS, DCM, rt. 2 h; c: HNO
3
,
,
3
3
, c H SO
2
4
, 1 h; f:
3
)
4
2
3
3
1
Scheme 1
14

Reagent and conditions: a: 1) ClSO
h; b: ClSO H, 0°C, 10 min; c: 3-amino-2,6-dimethoxypyridine, K
d: H , Pd/C, 3 h; e: CH
m-CPBA, DCM, 24 h; h: CH
3
H, DCM, 0°C to rt. 2 h; 2) SOCl
CO
, 5 h; f: NaBH , THF, 0.5 h ; g:
I, NaOH, DMF, 4 h.
2
, DMF, 80°C, 1
3
2
3
, DMF, rt. 1h;
2
3
OH/DCM, H
OH, H
2
O
2
, c H
2
SO
4
4
3
2
O
2
, c H
2
SO
4
, 24 h; i: CH
3
Scheme 2
15

Table 1
Antiproliferative activity of 13a-13m in HepG2 and Aqueous solubility.
a
b
IC50 (µM)
HepG2
0.26
Aqueous solubility (µg/mL)
Comp.
R
pH 2.0
pH 7.4
c
c
1
3a
3b
6-Br
-
-
1
6-NO
2
0.082
0.26
0.17
1.7
0.11
1.4
1
3c
3d
3e
3f
6-CHO
7-NO
1
2
>25
0.11
0.41
0.84
0.14
0.21
15.8
0.5
< 0.10
0.23
1.53
0.13
0.17
1.0
1
7-CHO
7-OH
0.22
1
0.012
0.051
7.22
1
3g
3h
7-CH
7-COOCH
7-NH
6-CH OH
6-OH
2
OH
1
3
1
3i
3j
2
0.070
0.11
1
2
1.3
13k
0.096
0.036
0.23
3.4
3.1
1
3l
6-NH
6-OCH
H
2
19.6
1.32
0.10
2.0
13m
3
0.31
< 0.10
3
0.095
0.003
0.002
d
d
Podo
CA-4
N
N
d
d
N
N
a
IC50 values, concentration required to inhibit 50% of human tumor cells proliferation
after 48 h treatment.
b
Compounds were measured at pH 7.4 and pH 2.0.
c “-”: No solution.
d
“N”: Not Tested.
16

Table 2
Data for 13b, 13f, 13g and 13i-13l against four tumor cells.
a
IC50 (µM)
MIA PaCa-
Comp.
R
MCF-
7
Bel-
7402
1.99
0.056
0.99
0.18
1.28
1.83
0.58
1.07
0.016
0.020
HepG2
2
1
3b
3f
3g
6-NO
2
0.082
0.012
0.051
0.070
0.11
0.18
0.051
2.38
0.092
0.91
0.43
0.29
0.18
0.012
0.003
0.36
0.014
0.69
0.036
0.87
0.45
0.42
0.68
0.020
0.005
1
7-OH
1
7-CH
2
OH
1
3i
3j
7-NH
2
1
6-CH
2
OH
13k
6-OH
0.096
0.036
0.095
0.003
0.002
13l
6-NH
2
3
H
Podo
CA-4
a
IC50 values, concentration required to inhibit 50% of human tumor cells proliferation
after 48 h treatment.
17

Graphical abstract
Novel Carbazole Sulfonamide Derivatives of Antitumor Agent: Synthesis,
Antiproliferative Activity and Aqueous Solubility
a
a
a
a*
a*
Lianqi Sun , Yanbin Wu , Yonghua Liu , Xiaofang Chen , Laixing Hu
Data of 13f, 13i and 3 against four tumor cells and aqueous solubility.
Aqueous solubility
(µg/mL)
IC50 (µM)
C
omp.
He
pG2
0.0
12
0.0
70
MIA
MC
F-7
0.0
14
0.0
36
0.6
Bel-740
2
pH
7.4
1.53
pH 2.0
PaCa-2
1
1
0.84
0
0
.051
.092
0.056
0.18
1.07
3f
15.8
0.10
1.0
3i
0.0
< 0.10
3
0.18
95
8
18